Field of Invention
This invention related to polarized light and, more particularly, to visualization and measurement of birefringent structures that possess molecular order or that are under strain.
Discussion of Related Art
Polarized light imaging of two-dimensional birefringence distribution is an established technique for analyzing the structure of various specimens. It can also be applied to study the vector or tensor fields associated with birefringence.
Polarized light has been used to obtain contrast in light microscopy. Various polarized light techniques have been developed for microscope studies of birefringence in biological unstained specimens, which caused by structural or internal anisotropy of the cell structure (S. Inoué, “A Method For Measuring Small Retardations of Structures in Living Cells”, Exp. Cell Res. 2, pp. 513-517, 1951; S. Inoué and K. R. Spring, Video Microscopy. The Fundamentals, 2nd ed., Plenum Press, New York, 1997; R. Oldenbourg and M. Shribak, “Microscopes”, in Handbook of Optics, Third Edition, Volume I: Geometrical and Physical Optics, Polarized Light, Components and Instruments, (ed. M. Bass), McGraw-Hill, New York, 2010).
Common arrangements of polarized light imaging microscope include use of a pair of crossed polarizers in the beam path, with one polarizer placed prior to the sample and one after it. The sensitivity of these methods is limited, and it is difficult to detect retardance is below 5 nm.
Also the contrast of conventional polarized light images is direction sensitive. It varies proportionally with sin2α, where α is the slow axis azimuth relatively to the direction of polarization of the illuminating light. If the specimen slow axis and the light polarization are parallel, the contrast is zero. The contrast is highest if the slow axis is oriented at 45° or 135°. It is therefore necessary to examine unknown objects at several azimuth orientations. In order to increase the contrast and remove the slow axis ambiguity we need to employ a polarization compensator introducing some ellipticity in the polarized light. In this case, the brightness is the maximal if a is 45°, and it is the minimal if α is 135° (N. H. Hartshorne, and A. Stuart, Crystals and the Polarizing Microscope, 4th ed., Edward Arnold, London, United Kingdom, 1970).
The orientation-independent birefringence imaging has been developing by many researcher groups around the world for two decades. However, most of the currently existing techniques relay on capturing several images in time sequence or simultaneously and complex digital image processing the raw data.
The first orientation-independent techniques were reported in 1992 by Otani (Proc. SPIE 1720, 346-354 (1992)) and Noguchi (Proc. SPIE 1720, 367-378 (1992)). They employed mechanically rotated waveplates.
A polarized light microscope, which contains a mechanically rotated linear polarizer and circular analyzer, was described by Glazier and Cosier in 1997 (A. M. Glazer, and J. Cosier, “Method and Apparatus For Indicating Optical Anisotropy,” UK Patent Application No. 2,310,925). Typically, six images of a specimen are taken while the linear polarizer is incremented in 30° steps; these images are then processed to yield the birefringence map, as described in an article (A. M. Glazer, J. G. Lewis, and W. Kaminsky, “An Automatic Optical Imaging System For Birefringent Media,” Proc. R. Soc. Lond. A 452, pp. 2751-2765, 1996). The microscope is not suitable for measuring low retardance specimens because it is strongly susceptible to light intensity variations, photon statistical noise, detector read-out noise, and digitization error.
In 1994 Oldenbourg and Mei replaced rotatable waveplates with variable liquid crystal waveplates and developed a method for measurement of birefringence distribution using three consecutive elliptical and one circular polarized state of the illumination beam (U.S. Pat. No. 5,521,705). This device is known as LC-polscope. In 2000 Shribak proposed several computation algorithms, which increase sensitivity and reduce measurement time (U.S. Pat. Nos. 7,202,950, 7,239,388, 7,372,567). Currently, Perkin Elmer (Waltham, Mass.) manufactures Oosight and Abrio, which employ the LC-polscope technique and Shribak's algorithms. The price of devices is about $20 k and $40 k accordingly.
Instead of generating the raw images sequentially, Shribak et al. proposed to generate them in parallel by using a non-polarizing beamsplitter with a set of polarization analyzers in the imaging path (Proc. SPIE 4819, 56-67 (2002), U.S. Pat. No. 7,079,247). Later Kaminsky received a patent on very similar system (U.S. Pat. No. 7,522,278). The simultaneous multiple imaging technique allows to avoid artifacts caused by movement organelles in live cells. But this approach require additional custom beam multiplicator with price $15 k, large chip CCD camera with price about $15 k, and special software for image aligning.
Birefringence measuring techniques with using return-path setup were proposed by Shribak (USSR patents 1210137, 1282202, 1290090, 1290091, 1390636, 1414097, 1431484; M. I. Shribak “Autocollimating Detectors of Birefringence”, in International Conference on Optical Inspection and Micromeasurements, Christophe Gorecki, Editors, Proc. SPIE 2782, pp. 805-813, 1996; and by M. I. Shribak, Y. Otani and T. Yoshizawa, “Return-Path Polarimeter For Two Dimensional Birefringence Distribution Measurement”, Polarization: Measurement, Analysis, and Remote Sensing II, Dennis H., Goldstein; and David B. Chenault; Eds. Proc., SPIE 3754, pp. 144-149, 1999).
The application of two-dimensional birefringence imaging to the analysis of inner stress in construction models using photoelasticity is also well known (Handbook on Experimental Mechanics, Ed. by Albert S. Kobayashi, Prentice Hall: Englewood Cliffs, 1987). E. A. Patterson and co-authors offered a full-field imaging polariscope (E. A. Patterson, W. Ji, and Z. Fwang, “On Image Analysis For Birefringence Measurements in Photoelasticity”, Optic Laser Engineering, 28, pp. 17-36, 1997). It has a circularly polarized illumination beam and six consecutive settings of an analyzer polarizer: left and right circular polarized settings and four linear polarized settings at 0°, 45°, 90° and 135°. The technique doesn't provide high sensitivity with low retardance specimens and use a polarization state analyzer comprising a mechanically rotated quarter waveplate and rotated linear analyzer.
While there have thus been shown various techniques for retardance measurement and two-dimensional retardance imaging, the existing techniques in the art require taking four or more readings; or are not well-suited to measurement of low-retardance samples; or do not operate with high speed; or offer less than adequate accuracy or noise.